The proposed project will continue our molecular genetic analysis of poliovirus protein processing. The approaches to be employed will take advantage of in vitro mutagenesis schemes for generating altered 3C polypeptides and altered precursor substrates for 3C activity. Mixed infections will be carried out with mutant polioviruses carrying phenotypically detectable 3C lesions to analyze the cis/trans natures of their defects. Mutant viruses bearing 3C lesions will be used in temperature-shift experiments to determine when, during the time course of an infection, protein processing and RNA synthesis are affected most. Based upon our initial success in generating viable proteinase mutants with double-stranded oligonucleotide cassettes, additional mutagenic casettes will be generated for the 3C proteinase. Such cassettes will be designed to potentially produce single and pairwise amino acid substitutions in 3C. In addition, single amino acid substitutions resulting from one cassette mutation will be cloned into 3C coding regions already containing a different amino acid substitution to determine if the second mutation is compensatory or merely exaggerates the processing phenotype of the original lesion. Chimeric 3C proteinases between coxsackievirus B3 and poliovirus type 1 will be generated. Using single-strand oligonucleotide mutagenesis to first generate convenient restriction endonuclease cleavage sites in genomic regions encoding both 3C polypeptides (without changing amino acids encoded), the proteinases will be divided up into a number of "domains" that can be easily manipulated to produce 3C chimeras. These chimeras will be analyzed in vitro for their abilities to cleave both polio and coxsackie substrates. The system employing in vitro translation of genetically defined polio transcripts will be exploited to produce amino acid insertions in specific structural domains of the P1 precursor to capsid proteins. The insertions will be assayed for their effects on the ability of the P1 polypeptide to serve as a substrate for 3C proteinase activity. The in vitro system will also be used to determine the precise P3 region genetic requirements for the production of 3C containing polypeptides that are capable of efficient cleavage of wild-type P1 polypeptides. The proposed studies will contribute to the elucidation of the regulatory sequences of the 3C proteinase and to the determination of how poliovirus uses these sequences to differentially interact with polypeptides destined for widely-different functions.